Methods and materials for the detection of dengue virus infection

ABSTRACT

The present invention provides monoclonal antibodies that are specific for the Dengue non-structural glycoprotein NS1 in monomeric and/or oligomeric (primarily dimeric) form, together with methods, including ELISA and lateral flow assays, that employ the disclosed antibodies for the early detection of Dengue virus infection. Diagnostic kits for the detection of Dengue infection are also provided, such kits including the disclosed monoclonal and/or polyclonal antibodies.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbers1R43AI074172-01 and 5R43AI074172-02 awarded by the National Institutesof Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the diagnosis of dengue virusinfection.

More specifically, the invention relates to the immunological detectionof dengue fever using monoclonal antibodies raised against the dengueglycoprotein NS1.

BACKGROUND OF THE INVENTION

Dengue fever is one of most threatening mosquito-borne viral diseases inhumans. It is caused by four closely related virus serotypes (DEN1,DEN2, DEN3 and DEN4) of the flavivirus genus within the Flaviviridaefamily. The dengue virus is transmitted by mosquitoes of the Aedes genus(particularly Aedes aegypti) and causes a febrile illness in tropicaland sub-tropical regions. More than two billion inhabitants live inendemic regions and are at risk of dengue virus infection. According tothe World Health Organization (WHO), the incidence of dengue infectionincreased 30-fold over the last 50 years (Dussart et al., Clin. VaccineImmunol., 2006, 13:1185-1189), and is responsible for an estimated50-100 million new infections annually, as well as 500,000hospitalizations and 30,000 deaths, mostly in children.

Clinical signs of dengue infection usually appear five to eight daysafter infection and are characterized by undifferentiated fever(referred to as dengue fever), accompanied by severe headaches, lumbago,muscle and joint pain, and shivering. From the third to the fifth day ofthe febrile phase, a congestive maculopapular rash may appear (referredto as conventional dengue). In the majority of cases, disease usuallysubsides within a week. However, in the severe form, the infection canprogress to hemorrhagic syndrome (referred to as dengue hemorrhagicfever) and fatal hypovolemic shock (referred to as dengue shocksyndrome), which has a high mortality rate.

All four dengue serotypes are able to cause clinical symptoms of dengueinfection, which provides a life-long immunity to the homologous dengueserotype, but does not protect against heterologous dengue serotype.Therefore, any person in an endemic area is susceptible to fourinfections during their lifetime. Secondary infection with aheterologous serotype may lead to more severe manifestations of disease,probably due to complement activation by antigen-antibody complexes(Young et al., J. Clin. Microbiol., 2000, 38:1053-1057; Xu et al., J.Clin. Microbiol., 2006, 44:2872-2878).

During the replication of dengue virus, a non-structural glycoprotein,NS1, associates with the membrane on the cell surface and is releasedinto the circulation as early as 1 day post-onset of symptoms (Xu etal., 2006, Ibid). The early expression of the NS1 protein makes it agood diagnostic target for an early dengue infection as antibody-baseddiagnostic assays are capable of detecting dengue-specific antibodyproduction later in the course of the infection (7-10 days postinfection).

The mature dengue NS1 protein contains 352 amino acid residues in a basepolypeptide of ˜40 kDa, with glycosylation increasing the apparent massof the protein on SDS-PAGE (Flamand et al., J. Virol., 1999,73:6104-6110). The protein includes twelve invariant cysteine residuesand two N-glycosylation sites (at N130 and N207) conserved among allflavivirus NS1 proteins, indicating their importance to the structureand function of the protein.

NS1 exists in both intracellular and extracellular forms. Immature NS1exists as a hydrophilic monomer in the endoplasmic reticulum lumen, andis rapidly processed into a stable hydrophobic non-covalent homodimer,with the subunits interacting via their carboxyl termini (Flamand etal., 1999, Ibid). Upon dimerization, NS1 becomes associated withintracellular membrane components. The ability to form intracellulardimers appears to be particularly important for trafficking andsecretion from the cell. In mammalian cells, NS1 is secreted frominfected cells into the extracellular milieu, either as a solubleprotein, which may be present in a higher oligomeric form than a dimer,or in association with microparticles but not with virions. Studies haveidentified NS1 soluble tetramers and soluble, detergent-labile, hexamers(Wallis et al., J. Biol. Chem., 2004, 279: 20729-20741; and Flamand etal., 1999, Ibid). It has been postulated that the glycosylation statusof NS1 determines the oligomeric distribution of secreted NS1. Eventhough the role of NS1 glycoprotein is not clearly defined, some studieshave indicated that intracellular NS1 glycoprotein may be indirectlyinvolved in viral replication, with extracellular NS1 glycoprotein beinginvolved in the formation of immunogenic complexes and triggeringcomplement mediated immune response, resulting in a more severe form ofillness.

Currently, there is no commercially available vaccine for dengue virus.In the absence of immunization, the monitoring of dengue virus outbreaksand serological mapping of new outbreaks become critically important tothe control and containment of infection. As clinical manifestations fordengue virus infections are quite unspecific, it is difficult to affirmdiagnosis without laboratory testing. Programs have been set up by WHOto actively monitor vector insects and cases of fever, as well as toperform serological and virological screening of individuals suspectedof being infected with dengue virus. Thus, the development of diagnosticassays for dengue infection is critically important.

Early diagnosis is essential for proper timely treatment of the patient.The currently available tests for dengue include RT-PCR for viral RNAand immunologic tests for dengue-specific antibody or viral proteins.However, many of these tests have significant disadvantages. Forexample, RT-PCR for viral RNA requires expensive laboratory equipmentand trained personnel, which makes it hard to use on a large scale or inrural areas. Some dengue-specific enzyme linked immunosorbent assays(ELISAs) can detect IgM or IgG that appear later during the course ofinfection, however diagnosis as early as day two of infection ispreferable (Alcon et al., J. Clin. Microbiol., 2002, 40:376-381).

A comparative analysis of four diagnostic methods for dengue infection,namely virus isolation, viral RNA detection, dengue specific IgMdetection and NS1 antigen detection, revealed that NS1 antigen detectionhad the highest sensitivity rate compared to the other three methods(Kumarasamy et al., Singapore Med. J., 2007, 48:669-673). Severalimmunological tests employing specific peptides (including NS1) derivedfrom dengue virus have been proposed. U.S. Pat. Nos. 7,282,341,6,870,032, 5,824,506, 6,682,883, 6,190,859 and PCT Patent PublicationWO99/009414 describe methods of using peptides as a diagnostic tool fordetermining the presence of dengue virus.

Alcon et al. (J. Clin. Microbiol., 2002, 40:376-381) have described anELISA for NS1 detection and demonstrated that NS1 is present at highlevels in patient sera during primary and secondary infection. NS1 isdetectable during the whole clinical phase of illness and can bedetected in the first few days of infection (as early as the first dayof fever). Falconar and Young have described the production ofdimer-specific and dengue virus group cross-reactive mouse monoclonalantibodies to dengue 2 virus NS1 (J. Gen. Virol., 1991, 72:961-965), andthe use of certain of these antibodies in an ELISA for NS1 has beendescribed (Young et al., J. Clin. Microbiol., 2000, 38:1053-1057). Highlevels of NS1 were found in acute phase sera, but not in convalescentphase sera, from some of the patients with serologically confirmeddengue 2 virus secondary infection.

U.S. Pat. No. 6,870,032 describes a method for detecting NS1 protein inthe hexameric form, and the selection of antibodies specific for NS1protein in hexameric form, together with the use of such antibodies inthe early detection of flavivirus infection. All these studiesdemonstrate that antibodies directed to different epitopes of NS1 andNS1 oligomeric forms, as well as NS1-immune complexes, may playdifferent roles in the diagnosis of dengue virus infection.

SUMMARY

Dengue virus NS1 is a versatile protein that exists in various forms.Recent reports have demonstrated that dengue virus NS1 can be expressedas a dimer and monomer in addition to oligomeric and hexamericconfigurations (Somnuke et al., Virology, 2011; 413(2):253-64). U.S.Pat. No. 6,870,032 discloses a NS1 detection assay based on recognitionof a hexamer form of the NS1 molecule. Herein we describe a set ofantibody reagents specific for dengue NS1 that recognize monomeric,dimeric and also possibly multimeric, forms of the mammalian expressedNS1.

The present disclosure provides monoclonal and polyclonal antibodies,and antigen binding fragments thereof, that are specific for monomericand oligomeric (primarily dimeric) forms of the dengue virus NS1polypeptide, together with methods for the use of such antibodies in thediagnosis of dengue virus infection, and kits for use in such methods.The disclosed methods and compositions are effective in the earlydetection of dengue virus infection and can readily be used in thefield, leading to rapid treatment and to control of disease outbreak.

In one aspect, polyclonal and monoclonal antibodies are provided thatare specific for the NS1 polypeptide originating from Dengue virus 2serotype and expressed as described in the U.S. Pat. No. 7,332,322 byFrolov et al., Feb. 19, 2008. The amino acid sequence of thispolypeptide is provided in SEQ ID NO: 1. In certain embodiments, themonoclonal antibodies disclosed herein specifically bind to anon-hexameric form of NS-1. In specific embodiments, the disclosedmonoclonal antibodies comprise at least one variable region selectedfrom the group consisting of amino acid sequences provided herein as SEQID NO: 2-8.

In related aspects, isolated polynucleotides that encode the disclosedamino acid sequences, together with vectors comprising suchpolynucleotides and host cells transformed or transfected with thepolynucleotides, are also provided.

In another aspect, the present disclosure provides diagnostic tests,including ELISA and lateral flow assays, employing the disclosedpolyclonal and/or monoclonal antibodies that can be effectively employedto detect dengue virus infection in a subject, such as a human. Suchassays can be employed to detect the presence of dengue virus infectionusing biological samples including blood, serum, plasma, saliva,cerebrospinal fluid, urine, and other tissue specimens (Monique da RochaQueiroz Lima et al., PLOSNTDS, v5 (5), e1147, 2011).

In one embodiment, the disclosed assays employ a first antibody specificfor dengue NS1 as a capture antibody, with a second antibody specificfor dengue NS1 in monomeric and/or oligomeric (primarily dimeric) formsbeing employed as a detection antibody. In certain embodiments, thesecond antibody is specific for a non-hexameric form of NS1. Preferably,the detection antibody is labeled with a detection agent. For example,in certain embodiments, a rabbit polyclonal antibody raised against NS1is employed as the capture antibody, and a monoclonal antibody disclosedherein is employed as the detection antibody. Alternatively, amonoclonal antibody disclosed herein can be employed as the captureantibody, with a rabbit polyclonal antibody raised against NS1 beingemployed as the detection antibody.

In certain embodiments, the detection reagent comprises a reporter groupselected from the group consisting of: enzymes, substrates, cofactors,inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups,colorimetric indicators and biotin. In the case of an ELISA, thedetection antibody can be unlabeled and a goat anti-mouse IgG1horseradish peroxidase can be employed as a separate detection reagent.Alternatively, the detection antibody can be conjugated to horseradishperoxidase prior to use in the assay. For a lateral flow assay, thedetection antibody is preferably labeled with a colorimetric orfluorescent indicator, such as colloidal gold or a fluorescent dye,thereby allowing a user to determine visually whether a test is positiveor negative for dengue infection.

In a related aspect, kits for the diagnosis of dengue virus infection byELISA are provided, such kits comprising: (a) a solid surface on which apolyclonal antibody specific for the dengue NS1 polypeptide isimmobilized; (b) at least one control selected from the group consistingof: positive dengue virus controls, negative dengue virus controls andcut-off controls; and (c) a solution containing at least one monoclonalantibody specific for a dengue NS1 polypeptide oligomer (primarilydimer) and/or monomer, as disclosed herein.

In a further aspect, dipsticks for use in the lateral flow assaysdisclosed herein are provided. In certain embodiments, such dipstickscomprise: (a) a lateral flow membrane; (b) a first area positioned at afirst, lower, end of the lateral flow membrane for receiving a testsample, wherein the first area comprises a first antibody specific for amonomeric and/or oligomeric (primarily dimeric) form of the dengue NS1polypeptide, the antibody being labeled with a reporter agent; (c) asecond area positioned at a second, upper, end of the lateral flowmembrane comprising an immobilized control polypeptide; and (d) a thirdarea positioned between the first and second areas, wherein the thirdarea comprises an immobilized second, different, antibody specific for amonomeric and/or oligomeric (primarily dimeric) form of the dengue NS1polypeptide. In certain embodiments, the first antibody is a polyclonalantibody, and the second antibody is a monoclonal antibody disclosedherein.

Diagnostic kits comprising such dipsticks are also provided. In certainembodiments, such diagnostic kits comprise a dipstick plus at least oneof the following components: a vessel containing lysing buffer; and apipette for applying the test solution to the dipstick.

In certain embodiments, the kits disclosed herein are sealably containedwithin a container, such as an aluminum pouch, that is generallyimpermeable to gases and fluids.

The above-mentioned and additional features of the present invention andthe manner of obtaining them will become apparent, and the inventionwill be best understood by reference to the following more detaileddescription. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a dipstick for use in a lateral flow assay of the presentdisclosure.

FIG. 2 shows the serotype specificity of dengue-2 NS1-specificmonoclonal antibodies of the present disclosure as determined using asandwich ELISA.

FIG. 3 shows the ability of the pan dengue specific mAb 528.292 incombination with the affinity purified rabbit anti-NS1 polyclonal seradisclosed herein to detect NS1 from all DENV serotypes using threedifferent assay formats.

FIG. 4 shows the ability of the pan dengue specific mAb 528.292 incombination with the affinity purified rabbit anti-NS1 polyclonal serato detect low levels of NS1.

FIG. 5 shows the ability of the pan dengue specific mAb 528.292 incombination with the affinity purified rabbit anti-NS1 polyclonal serato react with supernatant from the four dengue serotypes.

FIG. 6 shows results demonstrating that the mAb 528.292 disclosed hereinis approximately 2-3 times more sensitive in a sandwich ELISA assay thanthe known pan DENV serotype reactive NS1 antibody 7E11.

FIG. 7 shows the results of an epitope competition assay using mAb528.292 and 7E11.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides methods and materialsfor detecting dengue virus infection in a biological sample, such asblood, serum, plasma, saliva, cerebrospinal fluid and urine, taken fromindividuals, such as humans and/or other mammals, suspected of beinginfected with dengue virus. The presence of dengue virus infection isdetected using one or more of the assays described herein to determinethe presence or absence of the dengue NS1 protein in a biologicalsample. The amino acid sequence for dengue virus NS1 is provided in SEQID NO: 1. In certain embodiments, the disclosed assays employ monoclonalantibodies specific for monomeric and/or oligomeric (primarily dimeric)forms of NS1. In other embodiments, the disclosed assays employmonoclonal antibodies specific for monomeric and/or oligomeric(primarily dimeric) forms of NS1 in combination with polyclonalantibodies specific for dengue virus NS1 polypeptide.

The monoclonal antibodies of the present disclosure specifically orselectively bind to a monomeric and/or oligomeric (primarily dimeric)form of the dengue NS1 polypeptide, while not significantly bindingother components present in a test sample. As used herein, the term“antibodies specific for” a defined target molecule refer to antibodiesthat bind a target molecule with an affinity or K_(a) (i.e., anequilibrium association constant of a particular binding interactionwith units of 1/M) of, for example, greater than or equal to about 10⁵M⁻¹, 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹ or10¹³ M⁻¹. Alternatively, binding affinity may be defined as anequilibrium dissociation constant (K_(d)) of a particular bindinginteraction with units of M (e.g., 10⁻⁵ M to 10⁻¹³ M). Bindingaffinities of antibodies can be readily determined using techniques wellknown to those of skill in the art (see, e.g., Scatchard et al. (1949)Ann. N.Y. Acad. Sci. 51:660; U.S. Pat. Nos. 5,283,173; 5,468,614;Biacore® analysis; or the equivalent). In certain embodiments, thepolyclonal and/or monoclonal antibodies disclosed herein are preselectedby immunocapture on the dengue virus NS1 glycoprotein. In specificembodiments, the antibodies disclosed herein are monoclonal antibodieshaving variable regions that include at least one amino acid sequenceselected from the group consisting of: SEQ ID NO: 2-8.

Terms understood by those in the art as referring to antibody technologyare each given the meaning acquired in the art, unless expressly definedherein. For example, the terms “V_(L)” and “V_(H)” refer to the variablebinding region derived from an antibody light and heavy chain,respectively. The variable binding regions are made up of discrete,well-defined sub-regions known as “complementarity determining regions”(CDRs) and “framework regions” (FRs). The terms “C_(L)” and “C_(H)”refer to an “immunoglobulin constant region,” i.e., a constant regionderived from an antibody light or heavy chain, respectively, with thelatter region understood to be further divisible into C_(H1), C_(H2),C_(H3) and C_(H4) constant region domains, depending on the antibodyisotype (IgA, IgD, IgE, IgG, IgM) from which the region was derived. Aportion of the constant region domains makes up the Fc region (the“fragment crystallizable” region), which contains domains responsiblefor the effector functions of an immunoglobulin, such as ADCC(antibody-dependent cell-mediated cytotoxicity), ADCP(antibody-dependent cell-mediated phagocytosis), CDC(complement-dependent cytotoxicity) and complement fixation, binding toFc receptors, greater half-life in vivo relative to a polypeptidelacking an Fc region, and protein A binding. A polypeptide containing anFc region allows for dimerization or multimerization of the polypeptide.A “hinge region,” also referred to herein as a “linker,” is an aminoacid sequence interposed between and connecting the variable binding andconstant regions of a single chain of an antibody, which is known in theart as providing flexibility in the form of a hinge to antibodies orantibody-like molecules. Immunoglobulin structure and function arereviewed, for example, in Harlow et al., Eds., Antibodies: A LaboratoryManual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor,1988). An extensive introduction as well as detailed information aboutall aspects of recombinant antibody technology can be found in thetextbook Recombinant Antibodies (John Wiley & Sons, NY, 1999). Acomprehensive collection of detailed antibody engineering laboratoryprotocols can be found in R. Kontermann and S. Dübel, Eds., The AntibodyEngineering Lab Manual (Springer Verlag, Heidelberg/New York, 2000).

Antibodies to purified, recombinant or synthesized antigens can beprepared by any of a variety of techniques known to those of ordinaryskill in the art. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In one such technique, animmunogen comprising the antigenic polypeptide is initially injectedinto any of a wide variety of mammals (e.g., mice, rats, rabbits, sheepand goats). In this step, the antigen, such as NS1, may serve as theimmunogen without modification. Alternatively, particularly forrelatively short polypeptides, a superior immune response may beelicited if the polypeptide is joined to a carrier protein, such asbovine serum albumin or keyhole limpet hemocyanin. The immunogen isinjected into the animal host, preferably according to a predeterminedschedule incorporating one or more booster immunizations, and theanimals are bled periodically. Polyclonal antibodies specific for theantigen may then be purified from such antisera by, for example,affinity chromatography using the antigen coupled to a suitable solidsupport.

Monoclonal antibodies specific for the antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and tested for bindingactivity against the polypeptide. Hybridomas having high reactivity andspecificity are preferred.

Monoclonal antibodies can be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies can then be harvested from the ascites fluid orthe blood. Contaminants can be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation and extraction.

There are a variety of assay formats known to those of ordinary skill inthe art for using antibodies to detect an antigen in a sample which canbe effectively employed in the disclosed methods. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. In one such assay format, an antibody, such as a polyclonalNS1-specific antibody (referred to as the capture antibody) isimmobilized on a solid support (as described below) and contacted withthe sample to be tested. After removal of the unbound sample, a secondmonoclonal antibody or antibody mixture specific for NS1 (referred to asthe detection antibody(s)), which has been labeled with a reportergroup, may be added and used to detect bound antigen.

In an exemplary competitive assay, the sample is combined with either amonoclonal or polyclonal antibody against NS1, which has been labeledwith a suitable reporter group. The mixture of sample and antibody isthen combined with polypeptide antigen, such as NS1, immobilized on asuitable solid support. Antibody that has not bound to NS1 in the sampleis allowed to bind to the immobilized antigen, and the remainder of thesample and antibody is removed. The level of antibody bound to the solidsupport is inversely related to the level of antigen in the sample.Thus, a lower level of antibody bound to the solid support indicates thepresence of dengue virus infection in the sample. To determine thepresence or absence of dengue virus infection, the signal detected fromthe reporter group that remains bound to the solid support is generallycompared to a signal that corresponds to a predetermined cut-off value.Such cut-off values may generally be determined as described below. Anyof the reporter groups discussed below may be used to label theantibodies, and binding may be detected by any of a variety oftechniques appropriate for the reporter group employed.

In one embodiment, the disclosed assay involves the use of antibodyimmobilized on a solid support to bind to and remove the antigen fromthe sample. The bound antigen may then be detected using a detectionreagent that binds to the antigen/antibody complex and contains adetectable reporter group. Suitable detection reagents includeantibodies that bind to the antigen/antibody complex labeled with areporter group. Alternatively, a competitive assay may be utilized, inwhich an antibody that binds to the antigen is labeled with a reportergroup and allowed to bind to the immobilized antigen after incubation ofthe antibody with the sample. The extent to which components of thesample inhibit the binding of the labeled antibody to the antigen isindicative of the reactivity of the sample with the immobilizedantibody.

The solid support may be any solid material known to those of ordinaryskill in the art to which the antibody may be attached. For example, thesolid support may be a test well in a microtiter plate, or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc formed of glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681.

The antibody may be bound to, or immobilized on, the solid support usinga variety of techniques known to those in the art, which are amplydescribed in the patent and scientific literature. As used herein, theterm “bound” refers to both noncovalent association, such as adsorption,and covalent attachment (which may be a direct linkage between theantibody and functional groups on the support or may be a linkage by wayof a cross-linking agent). In certain embodiments, binding by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption can be achieved by contacting the antibody, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and 1 day. In general, contacting a well of a plastic microtiterplate (such as polystyrene or polyvinylchloride) with an amount ofantibody ranging from about 10 ng to about 1 μg, and preferably about100 ng, is sufficient to bind an adequate amount of antibody.Nitrocellulose will bind approximately 100 μg of protein per cm³.

Covalent attachment of the antibody to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the antibody. For example, the antibody maybe bound to supports having an appropriate polymer coating usingbenzoquinone or by condensation of an aldehyde group on the support withan amine and an active hydrogen on the antibody (see, e.g., PierceImmunotechnology Catalog and Handbook (1991) at A12-A13).

In certain embodiments, the assay is an enzyme linked immunosorbentassay (ELISA). This assay is performed by first immobilizing an antibody(referred to as the capture antibody) on a solid support, such as thewell of a microtiter plate. The immobilized antibody is then incubatedwith the biological sample, and antigen, such as NS1 (if present in thesample) is allowed to bind to the antibody, to form an antibody-antigencomplex or conjguate. The sample may be diluted with a suitable diluent,such as phosphate-buffered saline (PBS) prior to incubation. In general,an appropriate contact time (i.e., incubation time) is that period oftime that is sufficient to detect the presence of dengue NS1 proteinwithin a dengue virus-infected sample. Preferably, the contact time issufficient to achieve a level of binding that is at least 95% of thatachieved at equilibrium between bound and unbound antigen. Those ofordinary skill in the art will recognize that the time necessary toachieve equilibrium may be readily determined by assaying the level ofbinding that occurs over a period of time. At room temperature, anincubation time of about 30 minutes is generally sufficient.

Unbound sample can then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™ and detectionreagent is added to the solid support. An appropriate detection reagentis any compound that binds to the immobilized antibody-antigen complexand that can be detected by any of a variety of means known to those inthe art. Preferably, the detection reagent contains a binding agent(such as, for example, Protein A, Protein G, immunoglobulin, lectin oran antibody) conjugated to a reporter group. Preferred reporter groupsinclude enzymes (such as horseradish peroxidase), substrates, cofactors,inhibitors, dyes, radionuclides, luminescent groups, fluorescent groupsand biotin. The conjugation of binding agent to reporter group may beachieved using standard methods known to those of ordinary skill in theart. Common binding agents may also be purchased conjugated to a varietyof reporter groups from many sources (e.g., Zymed Laboratories, SanFrancisco, CA and Pierce, Rockford, Ill.).

The detection reagent is then incubated with the immobilizedantibody-antigen complex for an amount of time sufficient to detect thebound antigen. An appropriate amount of time may generally be determinedfrom the manufacturer's instructions or by assaying the level of bindingthat occurs over a period of time. Unbound detection reagent is thenremoved and bound detection reagent is detected using the reportergroup. The method employed for detecting the reporter group depends uponthe nature of the reporter group. For radioactive groups, scintillationcounting or autoradiographic methods are generally appropriate.Spectroscopic methods may be used to detect dyes, luminescent groups andfluorescent groups. Biotin may be detected using avidin, coupled to adifferent reporter group (commonly a radioactive or fluorescent group oran enzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of NS1 polypeptide in the sample,the signal detected from the reporter group that remains bound to thesolid support is generally compared to a signal that corresponds to apredetermined cut-off value. This cut-off value is preferably theaverage mean signal obtained when the immobilized antibody is incubatedwith samples from an uninfected patient. In general, a sample generatinga signal that is three standard deviations above the mean is consideredpositive for NS1 and dengue virus infection. In an alternate embodiment,the cut-off value is determined using a Receiver Operator Curve,according to the method of Sackett et al., Clinical Epidemiology: ABasic Science for Clinical Medicine, p. 106-7 (Little Brown and Co.,1985). Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (100%-specificity) that correspond to each possiblecut-off value for the diagnostic test result. The cut-off value on theplot that is the closest to the upper left-hand corner (i.e., the valuethat encloses the largest area) is the most accurate cut-off value, anda sample generating a signal that is higher than the cut-off valuedetermined by this method may be considered positive. Alternatively, thecut-off value may be shifted to the left along the plot, to minimize thefalse positive rate, or to the right, to minimize the false negativerate. In general, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive fordengue virus infection.

In one embodiment, the present disclosure provides an ELISA sandwichassay that can be effectively employed to detect the presence of denguevirus infection in a biological sample. In this assay, an antibodyspecific for a monomeric and/or oligomeric (primarily dimeric) form ofNS1 (referred to as the capture antibody) is coated onto ELISA plates.After blocking, the plates are incubated with the biological sample,washed and then incubated with a second antibody specific for amonomeric and/or oligomeric (primarily dimeric) form of NS1 (referred toas the detection antibody), prior to being developed. In one embodiment,the capture antibody is a purified rabbit polyclonal antibody that hasbeen affinity purified against NS1, the detection antibody is aNS1-specific monoclonal antibody disclosed herein, and the plate isdeveloped using a goat anti mouse IgG1 horseradish peroxidase conjugate.

In a second embodiment, the assay is performed in a flow-through orlateral flow format, wherein the anti-NS1 antibody is immobilized on amembrane such as nitrocellulose.

In the flow-through test, antigens within the sample bind to theimmobilized antibody as the sample passes through the membrane. Adetection reagent then binds to the antibody-antigen complex as asolution containing the detection reagent flows through the membrane.The detection of bound detection reagent may then be performed asdescribed above. In the lateral flow format, one end of the membrane towhich antibody is bound is immersed in a solution containing thebiological sample. The sample migrates along the membrane through aregion containing the detection reagent, which preferably includes acolorimetric label, such as colloidal gold, and to the area ofimmobilized capture antibody. Concentration of detection reagent at thecapture antibody indicates the presence of dengue NS1 in the sample.Such tests can typically be performed with a very small amount ofbiological sample.

FIG. 1 shows an exemplary dipstick which may be employed in theinventive methods to detect the presence of NS1 in a biological sample.In this system, lines are striped (1 μl/cm) on a suitable membrane usingan automated biojet system (BioDot, Inc., Irvine, Calif.). The bottom,or test, line 10 consists of a NS1-specific capture antibody in asuitable buffer. One or more test lines can be employed depending on thenumber of antibodies to be incorporated. The top, or control, line 12 isused as an internal control to make sure that all the test componentsare working. In this embodiment, control line 12 is protein G or goatanti mouse or rabbit IgG. An anti-NS1 antibody (“detection antibody”)conjugated to gold is employed as the detection reagent. Colloidal goldconsists of discrete, electron-dense, red-colored particles. Whenconcentrated on solid surfaces, these particles can be visuallyobserved. The detection reagent is dried onto a glass fiber pad, orconjugate pad, 14 and laminated below a membrane. Pads that can beeffectively employed as conjugate pad 14 include those available fromWhatman Inc. (Florham Park, N.J.), such as the Whatman Rapid ReleasePad. A sample pad 16 is soaked in an appropriate buffer, dried andlaminated underneath pad 14. Pads that may be effectively employed assample pad 16 include those available from Ahlstrom Inc. An absorbenttop pad 18 is provided to remove excess fluid.

In use, the biological sample is applied onto the end of sample pad 16and followed with a chase buffer, preferably a phosphate based buffer.The sample mixes with the detection reagent and the resulting complexmoves laterally upward and binds to the capture antibody at test line 10if the sample contains NS1, causing test line 10 to turn red. Unboundcomplex will continue to travel upwards and will bind to the protein Gor goat anti-mouse or rabbit IgG at control line 12 depending on thenature of the antibody chosen for the mobile phase, causing control line12 to turn red. If a control line is not observed, the test isconsidered invalid.

Other formats for using the antibodies disclosed herein to detect denguevirus infection in a sample will be apparent to those of ordinary skillin the art. For example, one of skill in the art will appreciate that amixture of monoclonal antibodies specific for the dengue virus NS1 canbe employed in place of a single NS1-specific monoclonal antibody.

The present disclosure also provides kits for use in the diagnosis ofdengue virus infection. In certain embodiments, such kits comprise: adipstick as described above preferably pouched in a generallyimpermeable container, such as an aluminum foil container, withdesiccant; a tube, or vessel, containing lysing buffer; and a disposabletransfer pipette for applying solution to the dipstick. In alternativeembodiments, such kits comprise: a solid surface, such as a microtiterplate with wells to which a polyclonal antibody specific for the denguevirus NS1 has been immobilized; and a solution containing at least onemonoclonal antibody disclosed herein. Such kits can further comprise: atleast one control sample selected from the group consisting of: negativecontrols, positive controls, and cut-off controls; and/or appropriatebuffers.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Preparation of Recombinant NS1 Glycoprotein

Full-length NS1 protein was prepared using a mammalian expressionsystem. Secreted NS1 was concentrated, purified using glasschromatography, and examined by SDS-PAGE under reducing conditions, withand without heat treatment. No heat treatment would ensure that NS1dimers can be observed by SDS-PAGE. The results demonstrated thepresence of glycosylated monomers, glycosylated dimers and a smallfraction of non-glycosylated monomers and non-glycosylated dimers.

EXAMPLE 2 Preparation of Antibodies to NS1 Glycoprotein

Polyclonal and monoclonal antibodies against the dengue NS1 glycoproteinisolated as described above were prepared as follows.

30 μg of purified NS1 (prepared as described above) in 50 μl of PBS wasemulsified with equal volume of Complete Freund's Adjuvant and injectedsubcutaneously into a 5 week old female Balb/c mouse. Seven days later,the same amount of NS1 emulsified with Incomplete Freund's Adjuvant wasinjected intraperitoneally into the same mouse. The injection wasrepeated once more 21 days after the initial injection. On days 42, 43and 46, the mouse received intraperitoneal injections of 30 μg of NS1 in50 μof PBS without adjuvant. Two days later the animal was sacrificed,and the spleen excised under sterile conditions.

The spleen was homogenized with scissors under serum free RPMI 1640medium, and passed through a nylon cell strainer to form a splenocytesuspension. Splenocytes were collected by centrifugation, erythrocytesremoved by Erythrocyte Lysis Reagent and washed with RPMI 1640. Mousemyeloma cells were grown in H-FSM medium, containing 5% FBS to a densityof 4.3×10⁵/ml. A total number of 5.6×10⁸ myeloma cells, and separatelysplenocytes, were washed extensively by centrifugation with RPMI 1640medium prewarmed to 37° C. The myeloma cells and splenocytes werecombined, centrifuged together, and the pellet was gently broken. Cellfusion was performed by adding polyethylene glycol (Mw 1,500) solutiondropwise to the cell pellet at 37° C. The resulting cells were washedwith RPMI 1640, resuspended in 300 ml of prewarmed H-SFM 5% FBS, anddistributed onto thirty 96-well cell culture plates, 100 μl of the cellsuspension per well.

The cells were allowed to grow for a day, and were selected with 130 μlper well of the double concentration of HAT medium in H-SFM with FBS.Seven days later, 130 μl of each well content was removed, and replacedwith similar, fresh medium, containing HAT at the recommendedconcentration.

Thirteen days after the cell fusion, culture supernatants of theresulting hybridoma lines were tested by ELISA. NS1 was coated ontoELISA plates (100 ng per well in 50 μl sodium carbonate/bicarbonatebuffer pH 9.6) overnight at 4° C., and the wells were blocked with 200μl per well of 5% nonfat dry milk in PBS containing 0.05% Tween™ 20(ELISA Wash Buffer, EWB) for 1 hour at room temperature. After blocking,the wells were washed six times with EWB in an automated plate washer,and the hybridoma culture supernatants were incubated in the wells for 1hour at room temperature. After another wash, the wells were incubatedwith goat antibodies specific for mouse immunoglobulin, labeled withhorseradish peroxidase for 1 hour at room temperature, washed again andvisualized with 3,3′,5,5′-tetramethylbenzidine reagent. The reaction wasstopped with 1N sulfuric acid and read on an ELISA plate reader at 450nm wavelength.

The hybridoma wells corresponding to positive ELISA results weretransferred into wells of a 24-well cell culture plate and grown in 1 mlof H-FSM /FBS medium, containing HT additive. The lines were repeatedlychecked for specific antibody secretion by ELISA, expanded, cloned outby limiting dilution and cryopreserved.

The amino acid sequences of the heavy chain variable regions ofmonoclonal antibodies 528.292, 528.1133 and 528.1299 are provided in SEQID NO: 2, 4 and 6, respectively, with the amino acids of the light chainvariable regions of monoclonal antibodies 528.292, 528.1133 and 528.1299being provided in SEQ ID NO: 3, 5 and 7, respectively. Subsequentre-sequencing of the heavy chain variable region of monoclonal antibody528.1299 gave the sequence of SEQ ID NO: 8.

The ability of the mAbs and rabbit polyclonal antibody to bind to dengueNS1 protein prepared as described above was examined by western blot.Purified NS1 protein samples, either treated or not treated by heat,were analyzed using the mAbs or rabbit polyclonal antibody as theprimary antibody. Either the anti-mouse-IgG-HRP or anti-rabbit-IgG-HRPconjugate was appropriately used as the secondary antibody followed byblot development with TMB reagent. The results demonstrated that bothNS1 monomers and NS1 dimers (glycosylated and non-glycosylated) arerecognized by mAbs 528 292 and 528 1299, and the rabbit polyclonal sera.In contrast, mAb 528 1133 appeared to recognize only the dimeric NSspecies and/or a heat-sensitive NS1 epitope.

EXAMPLE 3 Characterization of Antibodies Raised Against NS1

Affinity purified rabbit polyclonal antibodies to NS1 were coated ontoELISA plates (100 ng per well in 50 μl sodium carbonate/bicarbonatebuffer, pH 9.6) overnight at 4° C. The wells were blocked with 200 μlper well of 5% nonfat dry milk in EWB for 1 hour at room temperature,and washed as above. Cell culture supernatants, containing dengue -1,-2, -3 and -4 serotype NS1 were reacted with plate bound rabbitantibodies, followed by incubation with NS1 specific monoclonalantibodies, and developed with goat antibodies specific for mouseimmunoglobulin, labeled with horseradish peroxidase. The ELISA resultswere visualized as above.

As shown above in FIG. 2, the NS1-specific monoclonal antibodies (mAbs)exhibited at least four classes of specificities: monospecific todengue-2 (e.g. mAb 528.2123, 528.211, 528.121 and 528.112); all dengueserotype-specific (“pan specific”, e.g. mAb 528.292); dengue-1/dengue-2specific (e.g. mAb 528.181); and mAbs reactive to all dengue serotypes,but with non-uniform reactivity (e.g. 528.1131, 528.2191, 528.1131 or528.1141; reacting strongest to dengue-2, to a smaller extent todengue-3, and an even smaller extent to dengue-1 or dengue-4).

Isotypes of dengue-NS1-specific mAbs were evaluated by ELISA, performedon microplates with bound recombinant NS1, as described above. Goatantibodies specific for mouse immunoglobulin were in this case replacedwith antibodies specific to single mouse IgG isotypes (IgG1, IgG2a,IgG2b, IgG3, and IgM). Most of the mAbs fall into IgG1 class (see Table1 below).

TABLE 1 Isotypes of some dengue-2 NS1-specific mAbs mAb Isotype 528.2123IgG1 528.2191 IgG1 528.292 IgG1 528.1131 IgG1 528.1141 IgG1 528.211 IgG1528.121 IgG1 528.181 IgG1 528.112 IgG2b

Further studies were performed using the pan dengue specific mAb 528.292in combination with the affinity purified rabbit anti-NS1 polyclonalsera to assess its performance in detection of NS1 from all DENVserotypes. Initially three different assay formats were tried: directlinking of mAb with HRP using Lightning Link; (b) using a polyclonalantibody to mouse IgG conjugated to HRP; and (c) using a polyclonalantibody to mouse IgG1 conjugated to HRP. The data are shown in FIG. 3and show that the use of the anti-mouse IgG conjugate gave the mostsensitive assay when tested with NS1 from DENV2.

Closer inspection of the low end sensitivity of this assay indicatedthat levels of 20 pg were detectable (FIG. 4). The assay was used todetect the reactivity with the supernatants containing the dengueserotypes and all four were shown to be reactive (FIG. 5).

EXAMPLE 4 Epitope Comparison for NS1-Specific Monoclonal Antibody598.292

A pan DENV serotype reactive NS1 antibody (7E11) was obtained that wasgenerated in a different format. This antibody was an IgG2a. It wasobserved that mAb 598.292 could not sandwich with this one so weanticipated they hit a similar epitope. Initially we optimized bothantibodies in a sandwich ELISA with the affinity purified polyclonalanti-NS1 and demonstrated that the sandwich assay using the 528.292antibody was 2-3 times more sensitive than that using 7E11 (FIG. 6).

An epitope competition assay was then performed to determine if 7E11 and528.292 would compete with 7E11 biotinylated in a similar fashion.Plates were coated with 25 ng of NS1 from DENV 2 and then reacted with7E11 biotinylated in combination with 7E11 unconjugated or 528.292unconjugated. A monoclonal antibody to BopE, an unrelated protein, wasused as a control. As shown in FIG. 7, the inhibition curves were almostidentical, indicating that the antibodies are hitting the same epitope.

EXAMPLE 5 ELISA for the Detection of Dengue Virus Infection

A prototype Dengue NS1 sandwich ELISA produced under GMP was evaluatedas described below. The ELISA employed a rabbit anti-NS1 captureantibody and three different monoclonal antibodies (528.292, 528.1299and 528.1133) prepared as described above as secondary reagents. Goatanti-mouse IgG1-HRP was used for the conjugate step. A panel of positiveand negative samples was screened using both the Dengue NS1 ELISA andRT-PCR. As shown in Tables 2 and 3 below, the antibodies demonstratedpan dengue serotype NS1 activity

More specifically, Dengue virus was cultured in vero cells and serialtwo-fold titrations of the supernatants of the cultures were evaluatedby ELISA using a mixture of mAbs 528.292, 528.1299 and 528.1133 in a1.6/1.2/1.2 ug/mL dilution. The Dengue NS1 ELISA showed strongreactivity with all four Dengue subtypes in vero culture. This wasfurther confirmed by evaluating the reactivity of the ELISA with serumsamples that had been classified as Dengue-1, -2, -3 or -4 using RT-PCRas shown in Table 3.

TABLE 2 Evaluation of Dengue NS1 ELISA with serial dilutions of dengueserotype specific Vero supernatants Fold Dengue-1 Dengue-2 Dengue-3Dengue-4 Dilution Vero Sup Vero Sup Vero Sup Vero Sup  400X 3.002 3.0693.034 3.034  800X 2.839 2.996 2.996 2.866  1600X 2.889 2.932 2.781 2.227 3200X 2.774 2.468 2.084 1.54  6400X 2.037 1.584 1.207 0.892 12800X1.228 0.966 0.708 0.574 25600X 0.698 0.551 0.43 0.365 51200X 0.443 0.3560.309 0.275 102400X  0.25 0.228 0.186 0.174 204800X  0.189 0.188 0.1590.17

TABLE 3 Detection of NS1 in patients from all Dengue serotypes DengueNS1 Sample ID Day RT-PCR ELISA Result RT-PCR characterized samples*S9902929 1 D1(31/29/29) 1.765 DN9900615A 5 D3(32/29/27) 3.148 DN9900258A7 D3(33/33/40) 2.869 DN9900497A 5 D4(38/35/30) 2.052 DN9900364A 7 F/D(NoCt/No Ct)* 0.280 S9902959 1 D2(24/20/27) 0.091 S9903037 1 D2(24/23/26)0.128 S10001534 2 D2(31/28/27) 0.107 S9904911 1 D3(34/30/31) 0.069S10001780 2 D4(34/26/29) 0.450 S9902358 2 D4(35/24/41) 2.301 DN9900334A6 D4(No Ct/35/31) 1.239 S10001780B 8 ND* 0.095 CK 2 ND* 3.159 S100006302 D1(22/22/23) 2.786 S9903229 1 D3(27/23/27) 0.923 54 2 D4(32/23/19)2.096 DN9900436A 2 D4(39/33/25) 0.674 45 — D4(41/33/29) 1.234 NegativeSamples DN10000184A 3 — 0.084 S1002348 — — 0.088 S1002347 — — 0.077S1002345 — — 0.091 S1002346 — — 0.07 S1002344 — — 0.086 S1002343 — —0.071 S1002342 — — 0.082 S1002341 — — 0.082 S1002340 — — 0.086 InBiosCut-off 0.105 *These samples were also evaluated using Dengue IgM ELISA.The Dengue IgM ratios (Dengue antigen/normal cell antigen) for samplesDN9900364A, S10001780B and CK were 19.20 (positive), 5.22 (positive) and1.17 (negative).

The ELISA was run according to the kit insert. Samples greater than thecut-off sample OD value were considered positive and samples lower thanthe cut-off sample OD value were considered negative.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention.

SEQ ID NO: 1-8 are set out in the attached Sequence Listing. The codesfor polynucleotide and polypeptide sequences used in the attachedSequence Listing confirm to WIPO Standard ST.25 (1988), Appendix 2.

All references disclosed herein, including patent references andnon-patent references, are hereby incorporated by reference in theirentirety as if each was incorporated individually.

We claim:
 1. A monoclonal antibody, or antigen binding fragment thereof, that is specific for a dengue virus NS1 polypeptide, wherein the monoclonal antibody comprises at least one variable region selected from the group consisting of: SEQ ID NO: 2-8.
 2. An isolated polynucleotide comprising a nucleic acid that encodes an amino acid sequence selected from the group consisting of: SEQ ID NO: 2-8.
 3. A host cell comprising the isolated polynucleotide of claim
 2. 4. A method for detecting dengue virus infection in a biological sample, comprising: (a) contacting the biological sample with a monoclonal antibody of claim 1 to form a monoclonal antibody-NS1 polypeptide complex; and (b) contacting the monoclonal antibody-NS1 polypeptide complex with a detection reagent that binds to the complex, thereby detecting dengue virus infection in the biological sample.
 5. A method for detecting dengue virus infection in a biological sample, comprising: (a) contacting the biological sample with a polyclonal antibody specific for a dengue NS1 polypeptide to form a polyclonal antibody-NS1 polypeptide conjugate; (b) contacting the polyclonal antibody-NS1 polypeptide conjugate with a monoclonal antibody of claim 1 to form a polyclonal antibody-NS1 polypeptide-monoclonal antibody conjugate; and (c) detecting the presence of the polyclonal antibody-NS1 polypeptide-monoclonal antibody conjugate, thereby detecting dengue virus infection in the biological sample.
 6. The method of claim 5, wherein at least one of the monoclonal antibody and the polyclonal antibody is labeled with a detection reagent.
 7. The method of claim 6, wherein the detection reagent comprises a reporter group.
 8. The method of claim 7, wherein the reporter group is selected from the group consisting of: enzymes, substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, colorimetric indicators, and biotin.
 9. The method of claim 5, wherein the biological sample is selected from the group consisting of: blood, serum, plasma, saliva, cerebrospinal fluid, and urine.
 10. The method of claim 9, wherein the biological sample is a mammalian sample.
 11. The method of claim 5, wherein the polyclonal antibody is immobilized on a solid support.
 12. A method for detecting dengue virus infection in a biological sample, comprising: (a) contacting the biological sample with a monoclonal antibody of claim 1 to form a monoclonal antibody-NS1 polypeptide conjugate; (b) contacting the monoclonal antibody-NS1 polypeptide conjugate with a polyclonal antibody specific for a dengue NS1 polypeptide to form a monoclonal antibody-NS1 polypeptide-polyclonal antibody conjugate; and (c) detecting the presence of the monoclonal antibody-NS1 polypeptide-polyclonal antibody conjugate, thereby detecting dengue virus infection in the biological sample.
 13. The method of claim 12, wherein at least one of the monoclonal antibody and the polyclonal antibody is labeled with a detection reagent.
 14. The method of claim 13, wherein the detection reagent comprises a reporter group.
 15. The method of claim 14, wherein the reporter group is selected from the group consisting of: enzymes, substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, colorimetric indicators, and biotin.
 16. The method of claim 12, wherein the biological sample is selected from the group consisting of: blood, serum, plasma, saliva, cerebrospinal fluid, and urine.
 17. The method of claim 16, wherein the biological sample is a mammalian sample.
 18. A diagnostic kit for detecting dengue virus infection in a biological sample, comprising: (a) a monoclonal antibody of claim 1; and (b) a detection reagent.
 19. The kit of claim 18, wherein the detection reagent comprises a reporter group selected from the group consisting of: enzymes, substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, colorimetric indicators, and biotin.
 20. A kit for detecting dengue virus infection in a biological sample, comprising: (a) at least one solid surface on which a polyclonal antibody specific for a dengue NS1 polypeptide is immobilized; (b) at least one control selected from the group consisting of: positive dengue virus controls, negative dengue virus controls and cut-off controls; and (c) a solution comprising a monoclonal antibody of claim
 1. 21. A dipstick for detecting dengue virus infection in a biological sample, comprising: (a) a lateral flow membrane; (b) a first area positioned at a first, lower, end of the lateral flow membrane for receiving a test sample, wherein the first area comprises a polyclonal antibody specific for a dengue NS1 polypeptide, the polyclonal antibody being labeled with a reporter agent; (c) a second area positioned at a second, upper, end of the lateral flow membrane comprising an immobilized control polypeptide; and (d) a third area positioned between the first and second areas, wherein the third area comprises an immobilized monoclonal antibody of claim
 1. 22. A kit comprising the dipstick of claim
 21. 23. A method for detecting dengue virus infection in a biological sample, comprising: (a) contacting the biological sample with a monoclonal antibody that binds to a non-hexameric form of NS1 to form a monoclonal antibody-NS1 polypeptide complex; and (b) contacting the monoclonal antibody-NS1 polypeptide complex with a detection reagent that binds to the complex, thereby detecting dengue virus infection in the biological sample. 